3. Proposal Summary/Abstract The T-Cell Intracellular Antigen-1 (Tia1) protein is involved in stress response, translation regulation, and also in learning and memory. A 46 residue stretch of Tia1 has 63 % sequence identity to the domain of the functional prion Sup35 required for its fibrillation properties. Tia1 is also known to be present as a high order oligomer when functionally upregulated in vivo. The gene sequence for TIA1 contains three RNA recognition motifs and a prion-related domain. Biophysical techniques indicate that the protein forms oligomers under many conditions both in vivo and in vitro. We will characterize Tia1, including studies leading to the atomic resolution structure, with solid-state NMR and cryogenic transmission electron microscopy methods in order to provide restraints for modeling oligomer assembly and RNA binding. Ultimately, these studies are expected to aid in identifying ligands for therapy or diagnosis for the numerous pathologies and stress responses associated with Tia1. Preliminary data suggests that the RNA binding domains are folded even in the oligomeric state, suggesting the possibility that the high order oligomer is fully functional, despite the presence of a prion related domain. We will direct our initial efforts to obtaining NMR peak assignments and secondary structure. The secondary structure alone will provide compelling new information to test various hypotheses in the literature about Tia1, including mechanisms of RNA binding and protein oligomerization. Dynamic nuclear polarization (DNP) methods will be used alongside conventional SSNMR for assignment of the chemical shifts; DNP is expected to increase assignment confidence, reduce the analysis time, and allow for the study of more mobile and unstructured regions. To provide restraints for determining the full 3D structure, we will measure direct interatomic distances between key residues in Tia1 and between the protein and its ligands. We will use a selective isotope enrichment strategy that is not reliant on complete shift assignments. Electron microscopy methods after particle classification and multidimensional reconstruction will be used to characterize the morphology of Tia1, providing vital long distance information and particle shape restraints. These studies will culminate in a high resolution structure of Tia1. This project will proved extensive training opportunities in the biology and biochemistry of a protein in stress granules , cryogenic electron microcopy and polarization enhanced multidimensional SSNMR spectroscopy This training will enable me to apply structural biology techniques to many other significant and challenging biological problems. 1